Regulation of hydrogen peroxide production by brain mitochondria by calcium and Bax

Abnormal accumulation of Ca²⁺ and exposure to pro-apoptotic proteins, such as Bax, is believed to stimulate mitochondrial generation of reactive oxygen species (ROS) and contribute to neural cell death during acute ischemic and traumatic brain injury, and in neurodegenerative diseases, e.g. Parkinson's disease. However, the mechanism by which Ca²⁺ or apoptotic proteins stimulate mitochondrial ROS production is unclear. We used a sensitive fluorescent probe to compare the effects of Ca²⁺ on H₂O₂ emission by isolated rat brain mitochondria in the presence of physiological concentrations of ATP and Mg²⁺ and different respiratory substrates. In the absence of respiratory chain inhibitors, Ca²⁺ suppressed H₂O₂ generation and reduced the membrane potential of mitochondria oxidizing succinate, or glutamate plus malate. In the presence of the respiratory chain Complex I inhibitor rotenone, accumulation of Ca²⁺ stimutated H₂O₂ production by mitochondria oxidizing succinate, and this stimulation was associated with release of mitochondrial cytochrome c. In the presence of glutamate plus malate, or succinate, cytochrome c release and H₂O₂ formation were stimulated by human recombinant full-length Bax in the presence of a BH3 cell death domain peptide. These results indicate that in the presence of ATP and Mg²⁺, Ca²⁺ accumulation either inhibits or stimulates mitochondrial H₂O₂ production, depending on the respiratory substrate and the effect of Ca²⁺ on the mitochondrial membrane potential. Bax plus a BH3 domain peptide stimulate H₂O₂ production by brain mitochondria due to release of cytochrome c and this stimulation is insensitive to changes in membrane potential.